This project's ultimate objective is to develop safe broad-spectrum antiviral therapeutics/prophylactics, since there are currently relatively few therapeutics for viruses, and most which do exist are highly virus- specific or have undesirable side effects. The project's proven Double-stranded RNA Activated Caspase Oligomerizer (DRACO) approach selectively induces apoptosis (cell suicide) in cells containing any viral double-stranded RNA (dsRNA), rapidly killing infected cells without harming uninfected cells. We have previously created a protein DRACO that binds to the cellular procaspase-9 caspase recruitment domain (CARD) to induce apoptosis in the presence of dsRNA. We have demonstrated that this protein DRACO is nontoxic and effective against 15 different viruses in cells and 3 viruses in mice. We now propose to collaborate with the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB) at Harvard Medical School in order to develop high-throughput screening assays to identify small chemical molecules that would have similar properties to the large DRACO protein but would be easier to manufacture, store, and use. Our specific aims are to: 1. Develop a simple, reliable primary assay for procaspase-9 CARD binding activity suitable for high- throughput screening of small molecules. In consultation with the NSRB lab, we will develop assays that are fully compatible with high-throughput screening equipment and compound libraries. 2. Develop a counter-screening assay to distinguish molecules that bind to procaspase-9 CARD from molecules that bind to Apaf-1 CARD. This same counter-screening assay can be used to screen out molecules that nonspecifically bind to proteins. 3. Collaborate with the NSRB lab to conduct test runs of both the primary and counter-screening assays using their compound library and robotic high-throughput screening equipment. We will use the results of those test runs to iteratively optimize the assay reagents, conditions, and protocols. The proposed work should advance DRACOs toward ultimate utility as safe, broad-spectrum antiviral therapeutics, filling a large gap in existing therapeutics.